Methods for predicting pregnancy outcome in a subject by hCG assay

ABSTRACT

The present invention provides a method of predicting pregnancy outcome in a subject by determining the amount of an early pregnancy associated molecular isoform of hCG in a sample. The present invention further provides a method for determining the amount of early pregnancy associated molecular isoforms of human chorionic gonadotropin (hCG) in a sample. The present invention also provides a diagnostic kit for determining the amount of early pregnancy associated hCG in a sample. The present invention additionally provides an antibody which specifically binds to an early pregnancy associated molecular isoform of human chorionic gonadotropin. Finally, the present invention provides methods for detecting trophoblast or non-trophoblast malignancy in a sample.

This application is a continuation-in-part application of U.S. Ser. No.09/017,976, filed Feb. 3, 1998, the contents of which are herebyincorporated by reference into this application.

The invention disclosed herein was made with United States Governmentsupport under National Institutes of Health Grant Nos. NIEHS ES-07589and HD 15454. Accordingly, the U.S. Government has certain rights inthis invention.

BACKGROUND OF THE INVENTION

Throughout this application, various publications are referenced byauthor and date. Full citations for these publications may be foundlisted alphabetically at the end of the specification immediatelypreceding the claims. The disclosures of these publications in theirentireties are hereby incorporated by reference into this application inorder to more fully describe the state of the art.

Early pregnancy loss (EPL) is a widespread, but largely undiagnosedproblem. In order to adequately diagnose and develop treatments for EPLit is essential to be able to detect and measure the rate of occurrenceof EPL. This is critically important in epidemiological studies, some ofwhich are related to exposures to known or suspected reproductive toxinsin the workplace, in the environment or by personal use. These earlypregnancy losses are often not recognized by women or physicians and aredetected solely by the measurement of hCG in the urine at the timebetween implantation and expected menses. They are sometimes termed“chemical pregnancies” or “occult pregnancies” A landmarkepidemiological study established that the incidence of EPL was 22% in apopulation of healthy women attempting to conceive (Wilcox, A. J., etal., 1988). This investigation employed a very sensitive (0.01 ng/mlhCG) assay which detected only the intact hCG molecule with the uniquebeta subunit carboxyterminal peptide present.

There are multiple likely causes for EPL and clinical spontaneousabortion including genetic abnormality, immunological dysfunction,untreated infection or other unknown physiological problems. Inaddition, losses may be caused by failure of human chorionicgonadotropin (hCG) to induce adequate response at its target, the corpusluteum. This could result from inadequate hormonal potency. “Nicking” ofthe beta subunit in the loop 2 region of the molecule, specificallybetween residues 44-49, can reduce biopotency of hCG. Cleaved peptidebonds in this area of the molecule also exhibit reduced biopotency andreduced immunochemical recognition by monoclonal antibodies directed tothe heterodimeric hormone (Cole, L. A., et al., 1991a; Cole , L. A., etal., 1991b; Puisieux, A., et al., 1990; Nishimura, R., et al., 1988;Nishimura, R. T., et al., 1989). Nicked forms of hCG were examined aspossibly more prevalent in EPL situations and, at least in partresponsible, for early pregnancy loss. Unfortunately many of the reportsclaiming that substantial concentrations of nicked hCG are producedduring pregnancy, losses or successful pregnancies, are not accurate dueto faulty assumptions regarding assay specificity. Carbohydrate-modifiedhCG can also exhibit either reduced or enhanced biopotency. It is knownthat if the hCG has much reduced sialic acid content and itscarbohydrate chains terminate in galactose, much hCG would be removed bythe liver receptor for such altered glycoproteins (Braun, J. R., et al.,1996; Kawasaki, T. and G. Ashwell, 1996). The circulating life-time ofasialo hCG is reduced and its in vivo potency is thereby low. Othercarbohydrate changes also alter circulating half life; glycoproteinsterminating in sulfate-N-acetyl galactosamine are also extracted by aspecific liver receptor and have reduced circulating lifetime(Baenziger, J. U., 1994; Fiete, D., et al., 1991).

At least two factors affect increased potency of hCG. First, it is knownthat a larger Stoke's radius will decrease clearance through the kidneyglomerulus which generally clears proteins above an effective size of70,000 very slowly. The effective size of urinary-isolated hCG is justat this borderline reduced clearance size. Generally, extra sugarcontent makes the hydrated radius of glycoproteins larger. It has beenshown that by adding the hCG beta COOH-terminal peptide to hFSH or hLH,their circulating life-times greatly increased (Fares, F. A. et al.,1992; Matzuk, M. M., 1990). This addition was thought mostly due to thecarbohydrate content of that peptide rather than simply the extrapolypeptide size. Second, increased negative charge of a protein willprolong its circulating time because of decreased renal clearance(Chmielewski, C. 1992, Quadri, K. H., et al., 1994; Maack, T., et al.,1985). This increased negative charge can be due to extra sialic acid orother negative groups, including sulfate such as is present on hLH andon the pituitary form of hCG (Birken, S., et al., 1996b). Changes whichaffect signal transduction at the receptor may also affect biopotency ofhCG. It is known that deglycosylated hCG has much reduced receptorpotency (Ravindranath, N., et al., 1992; Sairam, M. R., and L. G.,Jiang, 1992; Browne, E. S., et al., 1990; Sairam, M. R., 1989; Sairam,M. R., et al., 1988). Carbohydrate reduced forms of hCG also havereduced signal transduction (Amano, J., et al., 1990; Bahl, O. P., etal., 1995; Moyle, W. R., 1975).

According to the present invention EPL or recurrent spontaneous abortionis not due to an abnormal hCG form that has reduced potency, such asnicked hCG. Instead, the present invention provides evidence that insuccessful outcome pregnancies women usually produce forms of hCG whichare very highly potent in very early pregnancy; the standard urinaryreference preparations of hCG are less potent forms of the hormoneproduced later in pregnancy. The increased potency could be caused by acombination of factors from circulating half-life to increased receptoraffinity or signal transduction or all of the preceding. Since hCG islow very early in pregnancy, it is logical to find a more potent form ofhCG on a molar basis to carry out its function until production levelsrise as the trophoblastic cellular mass increases. The present inventiondescribes molecular and immunological tools and methods including anantibody, B152, described herein which recognizes the highly potentearly pregnancy associated molecular isoforms of hCG. The determinationof blood and urine profiles for the B152 hCG isoforms throughout healthypregnancies can delineate the pattern of isoforms in successfulpregnancies. These isoforms can be measured by immunoassay alone,obviating the need to perform complex isoelectric focusing studies orother separation techniques. Additionally, the methods described hereinare applicable to large numbers of samples.

SUMMARY OF THE INVENTION

The present invention provides a method of predicting pregnancy outcomein a subject by determining the amount of an early pregnancy associatedmolecular isoform of hCG in a sample comprising: (a) contacting a samplewith an antibody which specifically binds to the early pregnancyassociated molecular isoform of hCG under conditions permittingformation of a complex between the antibody and the early pregnancyassociated molecular isoform of hCG; (b) measuring the amount ofcomplexes formed, thereby determining the amount of the early pregnancyassociated molecular isoform of hCG in the sample; and (c) comparing theamount early pregnancy associated molecular isoform of hCG in the sampledetermined in step (b) with the amount determined for temporallymatched, normal pregnant subject(s) wherein the relative absence of theearly pregnancy associated molecular isoform of hCG in the sampleindicates a negative outcome of pregnancy for the subject.

The present invention further provides a method of predicting pregnancyoutcome in a subject by determining the amount of an early pregnancyassociated molecular isoform of hCG in a sample comprising: (a)contacting a capturing antibody which specifically binds to the earlypregnancy associated molecular isoform of hCG with a solid matrix underconditions permitting binding of the antibody with the solid matrix; (b)contacting the bound matrix with the sample under conditions. permittingbinding of the antigen present in the sample with the capturingantibody; (c) separating the bound matrix and the sample; (d) contactingthe separated bound matrix with a detecting antibody which specificallybinds to hCG under conditions permitting binding of antibody and antigenin the sample; (e) measuring the amount of bound antibody on the boundmatrix, thereby determining the amount of early pregnancy associatedmolecular isoform of hCG in the sample; and (f) comparing the amountearly pregnancy associated molecular isoform of hCG in the sampledetermined in step (e) with the amount determined for temporallymatched, normal pregnant subject(s), wherein amounts of the earlypregnancy associated molecular isoform of hCG in the sample similar toamounts of early pregnancy associated molecular isoform of hCG intemporally matched pregnant samples indicates a positive outcome,amounts of early pregnancy associated molecular isoform of hCG in thesample similar to amounts of early pregnancy associated molecularisoform of hCG in the non-pregnant samples indicates a negative outcomeof pregnancy for the subject.

In addition, the present invention provides a method for determining theamount of early pregnancy associated molecular isoforms of in a samplecomprising: (a) contacting the sample with an antibody whichspecifically binds to an early pregnancy associated molecular isoform ofhCG under conditions permitting formation of a complex between theantibody and the early pregnancy associated molecular isoform of hCG;and (b) determining the amount of complexes formed thereby determiningthe amount of early pregnancy associated molecular isoform of hCG in thesample.

Further, the present invention provides a diagnostic kit for determiningthe amount of early pregnancy associated hCG is a sample comprising: (a)an antibody which specifically binds to an early pregnancy associatedmolecular isoform; (b) a solid matrix to which the antibody is bound;and (c) reagents permitting the formation of a complex between theantibody and a sample.

The present invention additionally provides an antibody whichspecifically binds to an early pregnancy associated molecular isoform ofhuman chorionic gonadotropin.

Further, the present invention provides a method for detectingnon-trophoblast malignancy in a sample comprising: (a) contacting asample with an antibody which specifically binds to the early pregnancyassociated molecular isoform of hCG under conditions permittingformation of a complex between the antibody and the early pregnancyassociated molecular isoform of hCG; (b) contacting the sample with asecond antibody which specifically binds to intact non-nicked hCGwithout substantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of. hCG; (c) measuring the amountof complexes formed, thereby determining the amount of the earlypregnancy associated molecular isoform of hCG in the. sample; and (d)comparing the amount of early pregnancy associated molecular isoform ofhCG in the sample determined in step (b) with the amount of earlypregnancy associated molecular isoform of hCG in the sample determinedin step (c), wherein a positive detection of early pregnancy associatedmolecular isoform detected in step (b) and a relative absence of theearly pregnancy associated molecular isoform of hCG detected in step (c)indicates the presence of non-trophoblast malignancy in the sample.

Finally, the present invention provides a method for detectinggestational trophoblast disease in a sample from a subject comprising(a) contacting a sample with an antibody which specifically binds to theearly pregnancy associated molecular isoform of hCG under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG; (b) contacting the samplewith a second antibody which specifically binds to intact non-nicked hCGwithout substantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG; (c) measuring the amountof complexes formed, thereby determining the amount of the earlypregnancy associated molecular isoform of hCG in the sample due tobinding with the first antibody, and late pregnancy associated molecularisoform of hCG in the sample due to binding with the second antibody;(d) determining the ratio of early pregnancy associated molecularisoform of hCG to late pregnancy associated molecular isoform of hCG inthe subject; and (e) comparing the ratio of early pregnancy associatedmolecular isoform of hCG to late pregnancy associated molecular isoformof hCG in the sample determined in step (c) over time, wherein acontinuing high ratio of early pregnancy associated molecular isoform ofhCG to late pregnancy associated molecular isoform of hCG in the sampledetermined in step (c) indicates the presence of gestational trophoblastdisease in the subject.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1.

Bioassay for forms of hCG. This is data from recombinant CHO cellsexpressing the LH/CG receptor. The response factor is cAMP production.The x-axis is dose of one of four calibrated, pure hormones as describedon graph legends. Expressed hCG has no nicks; choriocarcinoma hCG (C5)is 100% nicked; CR 127 was purified into a nick-free (intact) andnick-enriched fraction as shown.

FIG. 2.

Incidence (Panel A) and expression level (Panel B) of hCG-relatedmolecules in the positive samples for each of the analyses measured (Inearly normal pregnancy, n=214; EPL cycles, n=49; and negative cycles,n=297)

FIG. 3.

Binding curves for three hCG types in the B152-B207* assay (upper panel)and the B109-B108* assay (lower panel).

FIG. 4.

Ratio of hCG isoforms measured by the B152-B207* and B109-B108* assaysin normal pregnancy urine (n=103) at different gestational ages.(Regression curve and 95% confidence intervals are shown, r²=0.79) . Aninflection point in the curve occurs at approximately 29 weeks.

FIG. 5.

Box plot of the B152/B109 ratio for pregnancy matched serum/urine at 5-6weeks of gestational age (n=12); or at 36-39 weeks of gestational age(n=11) and in JAR cell supernatant. Box extends to the 25th and 75thpercentile. The upper and lower symbols indicate the 90th and 10thpercentile respectively. A solid line inside the box marks the value ofthe 50th percentile.

FIG. 6.

Ratio of hCG isoforms measured by the B152-B207* and B109-B108* assaysin the urine of IVF patients (n=65). (Regression curve and 95%confidence intervals are shown, r²=0.59).

FIG. 7.

Immunoassay profiles of fractions from Superose 12 column chromatographyof a pooled urine concentrate from pregnant women.

DETAILED DESCRIPTION OF THE INVENTION

A method of predicting pregnancy outcome in a subject by determining theamount of an early pregnancy associated molecular isoform of hCG in asample comprising: (a) contacting a sample with an antibody whichspecifically binds to the early pregnancy associated molecular isoformof hCG under conditions permitting formation of a complex between theantibody and the early pregnancy associated molecular isoform of hCG;(b) measuring the amount of complexes formed, thereby determining theamount of the early pregnancy associated molecular isoform of hCG in thesample; and (c) comparing the amount early pregnancy associatedmolecular isoform of is hCG in the sample determined in step (b) witheither (i) the amount determined for temporally matched, normal pregnantsubject(s) or (ii) the amount determined for non-pregnant subject (s),wherein the relative absence of the early pregnancy associated molecularisoform of hCG in the sample indicates a negative outcome of pregnancyfor the subject. In an embodiment of the present invention, the antibodyis B152. Another embodiment of this invention is the early pregnancyassociated molecular isoform of hCG.

The hybridoma producing the B152 monoclonal antibody was was depositedon Feb. 3, 1998 with the American Type Culture Collection (ATCC),. 12301Parklawn Drive, Rockville, Md. 20852, U.S.A. under the provisions of theBudapest Treaty for the International Recognition of the Deposit ofMicroorganism for the Purposes of Patent Procedure. The hybridoma, wasaccorded ATCC Accession Number HB-12467.

According to one embodiment of this invention, step (a) furthercomprises a second antibody which specifically binds to hCG withoutsubstantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG. In an embodiment of thisinvention, the second detection antibody is B207. According to anotherembodiment of this invention, step (a) further comprises a second assayantibody B109 which specifically binds to intact non-nicked hCG withoutsubstantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG. In an embodiment of thisinvention, the detection antibody is B108. In an embodiment of thisinvention, step (c) comprises comparing the amount of the earlypregnancy associated molecular isoform of hCG determined in step (b) forB152-B207 assay with the amount determined in step (b) for the B109-B108assay wherein a high ratio of amounts determined for said antibodyrelative to the second antibody indicates a positive outcome ofpregnancy for the subject, a low ratio indicates a negative outcome ofpregnancy for the subject.

In yet another embodiment of this invention, step (c) comprisescomparing the amount early pregnancy associated molecular isoform of hCGin the sample determined in step (b) with the amount determined fortemporally matched, normal pregnant subject(s), wherein amounts of theearly pregnancy associated molecular isoform of hCG in the samplesimilar to amounts of early pregnancy associated molecular isoform ofhCG in temporally matched pregnant samples indicates a positive outcome,amounts of early pregnancy associated molecular isoform of hCG in thesample similar to amounts of early pregnancy associated molecularisoform of hCG in the non-pregnant samples indicates a negative outcomeof pregnancy for the subject.

This invention also provides a method of predicting the likelihood of anegative pregnancy outcome in a female subject comprising: (a)contacting a sample from the subject with a capture antibody whichspecifically binds to an early pregnancy associated molecular isoform ofhCG under conditions permitting formation of a complex between theantibody and the early pregnancy associated molecular isoform of hCG;(b) contacting any complex formed in step (a) with a labelled detectionantibody under conditions permitting binding to the complex the captureantibody and the hCG isoform; (c) measuring the amount of labeleddetection antibody bound to the complex so as to thereby determine theamount of the early pregnancy associated molecular isoform of hCG in thesample; and (d) comparing the amount early pregnancy associatedmolecular isoform of hCG in the sample determined in step (b) with theamount determined for a normal pregnant subject, wherein the relativeabsence of the early pregnancy associated molecular isoform of hCG inthe sample indicates a negative outcome of pregnancy for the subject.

According to an embodiment of this invention, the sample is a urinarysample or a blood sample. In one embodiment of this invention, thesample is an aggregate sample taken from at least one day. In anotherembodiment, sample may be taken from at least two consecutive days andin a further embodiment, the sample is taken in three days. In anembodiment of this invention, the sample is a spot urine sample, a firstmorning void urine sample, or an aggregate sample of the first morningvoid urine samples for at least two consecutive days. In one embodimentof this invention, the antibody is labeled with a detectable marker. Inan embodiment of this invention, the detectable marker is a radioactiveisotope, enzyme, dye, magnetic bead, or biotin. In a preferredembodiment, the radioactive isotope is I¹²⁵.

The present invention further provides a method of predicting pregnancyoutcome in a subject by determining the amount of an early pregnancyassociated molecular isoform of hCG in a sample comprising: (a)contacting a capturing antibody which specifically binds to the earlypregnancy associated molecular isoform of hCG with a solid matrix underconditions permitting binding of the antibody with the solid matrix; (b)contacting the bound matrix with the sample under conditions permittingbinding of the antigen present in the sample with the capturingantibody; (c) separating the bound matrix and the sample; (d) contactingthe separated bound matrix with a detecting antibody which specificallybinds to hCG under conditions permitting binding of antibody and antigenin the sample; (e) measuring the amount of bound antibody on the boundmatrix, thereby determining the amount of early pregnancy associatedmolecular isoform of hCG in the sample; and (f) comparing the amountearly pregnancy associated molecular isoform of hCG in the sampledetermined in step (e) with the amount determined for temporallymatched, normal pregnant subject(s), wherein amounts of the earlypregnancy associated molecular isoform of hCG in the sample similar toamounts of early pregnancy associated molecular isoform of hCG intemporally matched pregnant samples indicates a positive outcome,amounts of early pregnancy associated molecular isoform of hCG in thesample similar to amounts of early pregnancy associated molecularisoform of hCG in the non-pregnant samples indicates a negative outcomeof pregnancy for the subject.

An embodiment of this invention further comprises (a) removing of thesample from the matrix; and (b) washing the bound matrix with anappropriate buffer. In one embodiment of this invention, the capturingantibody is B152. In one embodiment of this invention, the detectingantibody is B207. In an embodiment of this invention, step (a) furthercomprises a second capturing antibody which specifically binds to intactnon-nicked hCG without substantially cross-reacting with said antibodyunder conditions permitting formation of a complex between the antibodyand the early pregnancy associated molecular isoform of hCG. Accordingto an embodiment of this invention, the second capturing antibody isB109 and the second detection antibody is B108. In an embodiment of thisinvention, step (d) further comprises a second detecting antibody whichspecifically binds to hCG without substantially cross-reacting with saidantibody under conditions permitting formation of a complex between theantibody and the early pregnancy associated molecular isoform of hCG. Inan embodiment of this invention, step (f) comprises comparing the amountof the early pregnancy associated molecular isoform of hCG determined instep (e) for said antibody with the amount determined in step (b) forthe second antibody, wherein a high ratio of amounts determined for saidantibody relative to the second antibody indicates a positive outcome ofpregnancy for the subject, a low ratio indicates a negative outcome ofpregnancy for the subject.

According to an embodiment of this invention, the sample is a urinarysample or a blood sample.. In one embodiment of this invention, thesample is an aggregate sample taken from at least two consecutive days.In an embodiment of this invention, the sample is a spot urine sample, afirst morning void urine sample, or an aggregate sample of the firstmorning void urine samples for at least two consecutive days. In oneembodiment of this invention, the antibody is labeled with a detectablemarker. In an embodiment of this invention, the detectable marker is aradioactive isotope, enzyme, dye, magnetic bead, or biotin. In apreferred embodiment, the radioactive isotope is I¹²⁵.

In addition, the present invention provides a method for determining theamount of early pregnancy associated molecular isoforms of in a samplecomprising: (a) contacting the sample with an antibody whichspecifically binds to an early pregnancy associated molecular isoform ofhCG under conditions permitting formation of a complex between theantibody and the early pregnancy associated molecular isoform of hCG;and (b) determining the amount of complexes formed thereby determiningthe amount of early pregnancy associated molecular isoform of hCG in thesample.

According to an embodiment of this invention, the antibody specificallybinds a region of the early pregnancy associated molecular isoform ofhCG comprising a carbohydrate moiety. In one embodiment of thisinvention the antibody is produced by a hybridoma cell line. In oneembodiment of this invention the antibody is B152.

Further, the present invention provides a diagnostic kit for determiningthe amount of early pregnancy associated hCG is a sample comprising: (a)an antibody which specifically binds to an early pregnancy associatedmolecular isoform; (b) a solid matrix to which the antibody is bound;and (c) reagents permitting the formation of a complex between theantibody and a sample. In an embodiment of this invention, the antibodyis B109 or B152. An embodiment of this invention further comprisescontrol sample(s) normal pregnant sample(s), nonpregnant sample(s), ormale sample(s). The kit may also contain detection antibodies such asB207 or B108.

According to an embodiment of this invention, the sample is a urinarysample or a blood sample. In one embodiment of this invention, thesample is an aggregate sample taken from at least one or may be two orthree consecutive days. In an embodiment of this invention, the sampleis a spot urine sample, a first morning void urine sample, or anaggregate sample of the first morning void urine samples for at leastone, or may be two or three consecutive days. In one embodiment of thisinvention, the antibody is labeled with a detectable marker. In anembodiment of this invention, the detectable marker is a radioactiveisotope, enzyme, dye, magnetic bead, or biotin. In a preferredembodiment, the radioactive isotope is I¹²⁵.

The present invention additionally provides an antibody whichspecifically binds to an early pregnancy associated molecular isoform ofhuman chorionic gonadotropin.

In an embodiment of this invention, the antibody specifically binds to aregion of the early pregnancy associated molecular isoform of humanchorionic gonadotropin comprising a carbohydrate moiety. According toone embodiment of this invention, the monoclonal antibody is B152. In anembodiment of this invention, a hybridoma cell (ATCC Accession No.HB-12467) is provided capable of producing monoclonal antibody B152.Another embodiment of this invention is the early pregnancy associatedmolecular isoform of hCG recognized by the B152 monoclonal antibody.

Further, the present invention provides a method for detectingnon-trophoblast malignancy in a sample comprising: (a) contacting asample with an antibody which specifically binds to the early pregnancyassociated molecular isoform of hCG under conditions permittingformation of a complex between the antibody and the early pregnancyassociated molecular isoform of hCG; (b) contacting the sample with asecond antibody which specifically binds to intact non-nicked hCGwithout substantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG; (c) measuring the amountof complexes formed, thereby determining the amount of the earlypregnancy associated molecular isoform of hCG in the sample; and (d)comparing the amount of early pregnancy associated molecular isoform ofhCG in the sample determined in step (b) with the amount of earlypregnancy associated molecular isoform of hCG in the sample determinedin step (c), wherein a positive detection of early pregnancy associatedmolecular isoform detected in step (b) and a relative absence of theearly pregnancy associated molecular isoform of hCG detected in step (c)indicates the presence of non-trophoblast malignancy in the sample.

According to an embodiment of this invention, the antibody is B152 orB109. In an embodiment of this invention, the detection antibody is B207for B152 assay, B108 for B109 assay. In an embodiment of this invention,the non-trophoblast malignancy is ovarian malignancy or prostatemalignancy.

According to an embodiment of this invention, the sample is a urinarysample or a blood sample. In one embodiment of this invention, thesample is an aggregate sample taken from at least two consecutive days.In an embodiment of this invention, the sample is a spot urine sample, afirst morning void urine sample, or an aggregate sample of the firstmorning void urine samples for at least two consecutive days. In oneembodiment of this invention, the antibody is labeled with a detectablemarker. In an embodiment of this invention, the detectable marker is aradioactive isotope, enzyme, dye, magnetic bead, or biotin. In apreferred embodiment, the radioactive isotope is I¹²⁵.

Finally, the present invention provides a method for detectinggestational trophoblast disease in a sample from a subject comprising(a) contacting a sample with an antibody which specifically binds to theearly pregnancy associated molecular isoform of hCG under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG; (b) contacting the samplewith a second antibody which specifically binds to intact non-nicked hCGwithout substantially cross-reacting with said antibody under conditionspermitting formation of a complex between the antibody and the earlypregnancy associated molecular isoform of hCG; (c) measuring the amountof complexes formed, thereby determining the amount of the earlypregnancy associated molecular isoform of hCG in the sample due tobinding with the first antibody, and late pregnancy associated molecularisoform of hCG in the sample due to binding with the second antibody;(d) determining the ratio of early pregnancy associated molecularisoform of hCG to late pregnancy associated molecular isoform of hCG inthe subject; and (e) comparing the ratio of early pregnancy associatedmolecular isoform of hCG to late pregnancy associated molecular isoformof hCG in the sample determined in step (c) over time, wherein acontinuing high ratio of early pregnancy associated molecular isoform ofhCG to late pregnancy associated molecular isoform of hCG in the sampledetermined in step (c) indicates the presence of gestational trophoblastdisease in the subject.

In an embodiment of this invention, the antibody is B152 or B109. Inanother embodiment of this invention, the detection antibody is B108 forB109 assay, B207 for B152 assay. In an embodiment of the presentinvention, the gestational trophoblast disease is choriocarcinoma orhydatidiform mole.

According to an embodiment of this invention, the sample is a urinarysample or a blood sample. In one embodiment of this invention, thesample is an aggregate sample taken from at least two consecutive days.In an embodiment of this invention, the sample is a spot urine sample, afirst morning void urine sample, or an aggregate sample of the firstmorning void urine samples for at least two consecutive days. In oneembodiment of this invention, the detection antibody B207 or B108 islabeled with a detectable marker. In an embodiment of this invention,the detectable marker is a radioactive isotope, enzyme, dye, magneticbead, or biotin. In a preferred embodiment, the radioactive isotope isI¹²⁵.

As described herein below, unexpected isoforms of hCG are producedduring normal early pregnancy. Using an in vitro bioassay, it appearsthat these isoforms have enhanced potency for signal transduction. Theseisoforms can be measured using the novel sensitive, immunoassaydescribed herein. This can help predict pregnancy outcome where onecause of early pregnancy loss is failure to produce the isoform of hCGof higher potency produced by successful pregnancies. This enablesphysicians to intervene to sustain a failing pregnancy. Identificationof the nature of the hCG isoform required might provide the properreagent needed to sustain pregnancy.

New antibodies for measurement of nicked forms of hCG described hereinbelow were developed based on the hypothesis that forms of hCG, whichhave greatly reduced bioactivity, contribute to early pregnancy loss(EPL), due at least in part to diminished biopotency. Evidence was foundthat the hCG that appears in EPL patients displays reduced biologicalactivity. However, it was determined that the cause of the reducedbioactivity is not the presence of nicked hCG in EPL patients. Instead,the hypothesis is that patients that carry pregnancies forward producean isoform of hCG with enhanced bioactivity. The instant inventiondescribes a unique immunochemical assay to measure this unexpected andpreviously un-characterized isoform of early pregnancy hCG directly inclinical samples of blood and urine. One of the antibodies developedreacted against a nicked form of hCG isolated from a choriocarcinomapatient, was not specific for a nicked form of hCG but appeared todiscriminate among carbohydrate variants of hCG. This antibody,designated B152, appears to preferentially bind hCG forms fromchoriocarcinoma patients. In studying the content of hCG isoforms duringpregnancy, the unique and unexpected observation was made that B152 inthe first four weeks of pregnancy measured much higher quantities of anisoform of hCG as compared to the standard hCG isoforms measured by theusual heterodimeric hCG assays exemplified by a previously describedB109 based assay. In fact, in early pregnancy (days 9,10,11postovulation) B152 measured as much as 20-fold more hCG, than didanother monoclonal antibody, B109. Later in pregnancy, the B152 isoformdeclines and is lower in third trimester pregnancy urine than thestandard isoforms measured by B109. A further striking observation wasthat in very early pregnancy, a high B152/B109 ratio correlates with asuccessful pregnancy outcome while a low ratio correlated with pregnancyloss. This discovery is important as the potentially overlooked isoformsof hCG described herein during pregnancy may be predictors of successfulpregnancy outcome. Such an assay has wide medical applications andprovides a clinician with opportunity to intervene very early inpregnancy if the assay indicated that the pregnancy appeared troubled.

An antibody, designated B152, produced by the hybridoma cell accordedATCC Accession number HB-12467 generated against a nicked form of hCGisolated from a choriocarcinoma patient, but not specific for nickedisoform hCG is able to discriminate among carbohydrate variants of hCG.B152 is specific for an early pregnancy associated molecular isoform ofhCG which in the first four weeks of pregnancy is measured at muchhigher quantities than the hCG standard isoforms measured by the usualheterodimeric hCG assays exemplified by a previously described B109based assay. Later in pregnancy, the B152 isoform declines and is lowerin third trimester pregnancy urine than the standard isoforms measuredby B109.

This invention is illustrated in the Experimental Details section whichfollows. These sections are set forth to aid in an understanding of theinvention but are not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which followthereafter.

EXPERIMENTAL DETAILS EXAMPLE 1 Analysis of Molecular Isoforms of hCG inEarly Pregnancy and Early Pregnancy Loss

Introduction

Almost all investigations of the incidence of early pregnancy loss(EPL), either in normal populations or in populations at risk as aconsequence of exposure to putative reproductive toxins (Hakim, R. B.,et al., 1995; Lasley, B. L., et al., 1995) use assays for heterodimeric,non-nicked hCG or combination assays which additionally include freebeta subunit and beta core fragment of hCG. One concern about the formsof hCG to include in the measurement in EPL was heightened with respectto the nicking phenomenon described above. Because nicked hCG moleculesare not measured by the antibodies employed in most EPL studies, theincidence of EPL is presumably underestimated by an amount proportionalto the extent of nicking in the urinary molecule. Another concern ofsignificant importance was a determination of the nature of the “hCGlike” immunoreactivity in the urine in the periovulatory surge of themenstrual cycle (O'Connor J., et al., 1995). Recent reports haveconfirmed the existence of and documented the structure of a sulfatedform of hCG produced in the pituitary (Birken, S., et al., 1996b). Thereis a pulsatile secretion of hCG in both men and non-pregnant women.(Odell, W. D.; Griffin, J., 1989 and Odell, W. D.; Griffin, J., 1987).The presence of a non-pregnancy associated form of sulfated hCG ofpituitary. origin, peaking at ovulation and perhaps persisting into theluteal phase, could potentially interfere with the accurate estimationof EPL.

Unappreciated isoforms of hCG in blood and urine very early in pregnancymay be more potent in vivo than the forms of hCG produced later inpregnancy. The absence of such isoforms may be one cause of earlypregnancy loss. A sensitive and specific immunoassay system was designedand made to measure unique early pregnancy associated molecular isoforms(EPMI) of hCG. These isoforms, likely to differ by carbohydratecomposition, are predictive of a successful pregnancy outcome. Whenthese early pregnancy associated molecular isoforms of hCG are absent orpresent in low concentration, the pregnancy may be lost very early andbe observed as only a “chemical” pregnancy. These hCG isoforms mayresemble the forms of hCG produced in some choriocarcinoma patients fromwhich the immunogen used to produce monoclonal antibody B152 was derivedas described herein below. The isoforms resemble those fromtrophoblastic disease not in terms of nicking or intact peptide chainsbut likely in carbohydrate content. The present invention describes thatthe molar ratio of B152 to B109 epitopes are predictive of a successfulpregnancy or a loss. Three categories of pregnant patients wereanalyzed: (a) normal pregnant women, (b) women who experience recurrentabortions, (c) women undergoing embryo implantation.

It is possible to determine the hCG isoforms present in the blood andurine of women who have a history of recurrent spontaneous abortion anda similar analysis of women undergoing embryo implantation. The combinedEPL and spontaneous abortion rate in healthy populations is 31%.Subjects who experience three consecutive recurrent spontaneousabortions have a 32% risk of sustaining another (Hill, J. A.; Anderson,D. J., 1990). In in vitro fertilization IVF pregnancy, the loss rate is70% with non-donor sperm and 50% when donor sperm is used. Delineationof pregnancies with a negative outcome from pregnancies with a positiveoutcome can be based on differences in the concentrations of EPMI hCGisoforms (i.e. as differences in the B152/B109 ratio in patients). Inaddition, specimens from gestational trophoblastic disease (GTD) can beused to discriminate between GTD and normal pregnancy.

Results

In vitro Bioassay for hLH/hCG

An hCG bioassay was constructed employing CHO cells expressingfunctional human LH/CG receptor. FIG. 1 illustrates the differences invitro in biological activity between nicked and non-nicked hCG asmeasured by this assay. This system, has been used to evaluate theactivity of pituitary and placental hCG (Birken, S., et al., 1996b).Preparations of hCG were tested for nicked and non-nicked molecularisoforms of hCG in a second recombinant bioassay system (Ho, H-H., etal., 1997). Similar results were obtained in both systems.

Normal Pregnancy Values Compared With EPL Values.

FIG. 2 indicated that nicked hCG is not a significant molar constituentof either early pregnancy or EPL. Data indicated that biologicalactivity is not correlated with nicked hCG, but is instead ascribed to aform of hCG recognized by the B152 monoclonal antibody—an earlypregnancy associated molecular isoform of hCG (EPMI hCG). It has beenestablished that there is diminished hCG bioactivity associated with EPLas compared to early normal pregnancy (Ho, H-H., et al., 1997). Thus,diminished hCG biological activity is a factor in EPL as a consequenceof a heretofore unappreciated isoform of hCG—an early pregnancyassociated molecular isoform of hCG. hCG Urinary Analytes. Metabolitesof hCG and hLH were studied in a variety of states (Birken, S., et al.,1996a). One study indicated a 31% pregnancy loss (Zinaman, M J, et al.,1996) while another indicated a 17.4% rate of early pregnancy loss basedon hCG assays (Ellish, N. J., et al., 1996). It is known that hCG andhCG beta core can be readily transferred from the uterus to thecirculation even in the absence of implantation (Chang, P. L., 1997).The molecular spectrum of hCG urinary analytes in EPL cycles, normalconceptive cycles and non-conceptive cycles has been evaluated. Thestudy design and demographics of the investigation have been described(Ellish, N. J., et al., 1996).

Briefly, three urine specimens per cycle, corresponding to days 9,10,11, post calculated day of ovulation were collected and analyzed in ascreening assay (the “combo”) which simultaneously detects intact,non-nicked hCG, hCG free beta subunit, and hCG beta core fragment.Individual determinations for each of these analytes, as well as fornicked hCG, and the form of intact hCG detected by monoclonal antibodyB152 (EPMI hCG) were performed on these specimens. In addition, sincethe concentration of luteal phase hLH urinary analytes is a concernbecause of cross-reaction in hCG assays, levels of intact hLH, hLH freebeta subunit and hLH beta core fragment were determined in the normalpregnancy cycles and the non-conceptive cycles. Table I summarizes thecharacteristics of immunometric assays employed. TABLE 1 Assay formatand specificity Intra- Inter- Assay % cross-reactivity assay assayformat Primary analyte with related analytes cv, % cv, % B109-B108*intact non-nicked <1%^(b) 6 12 hCG B201-C104* hCG free beta 1% hCG; 6 12subunit (non- 10% hCG nicked nicked + nicked) (pregnancy); <1%^(b)B210-B108* hCG beta core 2% hLH beta core 5 7 fragment fragment; <1%^(b)B151-B207* hCG nicked 10% hCG nicked 5 15 free beta subunit; 12% hCGnon-nicked; 2% hCG free beta subunit; 2% hLH; 5% hLH free beta subunit;<1%^(b) B152-B207* choriocarcinoma 100% hCG nicked (C5); 6 13 hCG (C5)and 190% hCG free beta choriocarcinoma nicked (from C5); hCG 10% hCGnicked free beta subunit (pregnancy); 5% hCG free beta nicked(pregnancy); 7% hCG (pregnancy); 6% hCG free beta subunit; <1%^(b)B406-A201* hLH <1%^(a) 4 10 B505-B503* hLH beta core <1%^(a) 9 9fragment B408-B409* hLH free beta 29% hLH; <1%^(a) 7 11 subunit^(a)(if not indicated) hLH, free beta hLH, hLH beta core fragment, hCG,free beta hCG, hCG beta core fragment;^(b)(if not indicated) the same as^(a) plus nicked hCG and nicked freebeta hCG (pregnancy).

The results indicate that nicked hCG does not constitute a significantmole fraction of urinary hCG immunoreactivity in either EPL or earlynormal pregnancy. In addition, there is a substantial excretion of hCGfree beta subunit in some subjects in both pregnancy and EPL. Further,both EPL and normal pregnancy cycles variably express all of themeasured analytes. Although both the incidence and level of expressionare different between EPL's and normal pregnancy, there is no hCGrelated analyte unique to either state. There was, however, a cleardifference between the hLH associated analytes in the control population(non-conceptive cycles) and the normal pregnancy group. Virtually all ofthe non-pregnancy cycles expressed hLH free beta subunit and hLH betacore fragment while only a third of the conceptive cycles had detectablelevels of either analyte. Intact hLH proved to be a minor constituent ofthe hLH profile in both groups.

These findings demonstrate both the necessity of measuring hCG beta corefragment in the detection of EPL, and also of making sure that the hCGbeta core assay does not cross- react with beta core hLH, which isdemonstrated to be present in that part of the luteal phase where EPLmeasurements are performed. The data is summarized in FIG. 2.

Statistical analysis was performed after transformation of analytevalues to mole fractions so as to produce a more useful analysis due tothe wide excursion of hCG analyte values among groups. The mole fractiondata were evaluated by discriminant analysis and by a mixed effectsmodel incorporating LMP. The discriminant analysis was performed bothwith and without “outliers” (defined as values greater than two standarddeviation from the mean) removed. Both approaches produced similarresults.

A quadratic discriminant analysis based on a cross-validation method inorder to minimize bias correctly classified 91% of the normal pregnancysubjects and 80% of the EPL subjects.

The mixed effects analysis, testing for interactions between molefraction of analyte and time since LMP found no significant time orgroup (EPL vs. normal) effects in the intact hCG assay. In the free betasubunit of hCG assay, there is a significant group effect but no timetrend. In both the hCG beta core fragment measurement and the B152measurement, both the hormone levels and the time trend from LMP weresignificantly different between the EPL and pregnancy groups. This studyproduced several important findings. It defined the spectrum of analyteswhich in both early pregnancy and EPL, thereby resolving the issue ofwhich hCG analytes to measure in epidemiological studies in which EPL isthe end point determination. More importantly, it illustrated for thefirst time that there are significant differences both in the pattern ofanalytes and the time course of their appearance between early normalpregnancy and EPL. This observation facilitates very early prediction ofa distressed pregnancy by urinary hCG measurements at a time which wouldpermit therapeutic intervention.

Immunoreactivity of Different Forms of hCG in the Two IRMA's (B152-B207and B109-B108)

The relative binding of three different forms of hCG (urinary hCG,pituitary hCG and choriocarcinoma hCG CS) has been characterized in thetwo hCG assays (FIG. 3). Urinary non-nicked hCG and pituitary hCG arerecognized nearly equally well by the two IRMASs, while CS recognitionis quite different. The B152-B207* assay is more sensitive to C5, whichis to be expected because B152 antibody was developed and selected onthe basis of higher affinity to C5. Urinary non-nicked hCG is purifiedfrom the CR127 preparation of pooled normal pregnancy hCG. Conversely C5is recognized with lower affinity by the B109-B108* assay, which hasprimary specificity for the hCG isoforms of later pregnancy.

We have developed a method to directly profile changes of hCG isoformsin serum or urine throughout pregnancy. Two IRMAs for hCG are employed,each based on monoclonal antibodies to different hCG epitopes. TheB109-B108* assay is a commonly used intact hCG assay to theheterodimeric-dependent epitope. A new assay, B152-B207*, is most likelysensitive to the carbohydrate portion of hCG carboxyterminal peptide.The same standard non-nicked hCG was used in both assays. Non-nicked hCGwas employed since the B109 assay reacts poorly with nicked forms of hCGwhile the B152 assay does not discriminate between nicked and non-nickedforms of the hormone. The B152 assay detected with greatly enhancedsensitivity hCG isoforms which appear earlier in pregnancy than isoformsmeasured by the B109 assay (O'Connor et al. 1998). Prior to developmentof the new immunometric assay system described in this report, it wasnot possible to readily discern the changes in hCG isoforms from veryearly pregnancy to mid pregnancy. The only available procedure forexamining these changes was isoelectric focusing of every patientspecimen followed by immunoassay of every focused fraction (Berger etal. 1993; Ulloa-Aguirre et al. 1990). The IEF pattern reflects theheterogeneity of the charged sugar, sialic acid which varies with themulti-antennary structures of the carbohydrate moieties in which sialicacid is the terminal sugar. Although we do not yet know the precisenature of the isoform epitopes being measured, the evidence forcarbohydrate discrimination is based upon the hyperglycosylatedstructure of the immunogen, C5, used to develop the B152 monoclonalantibody and the antibody's reactivity with the hCG isoforms found inthe JAR choriocarcinoma cell line. C5 hCG was isolated from achoriocarcinoma patient and has been thoroughly characterized as to itsprotein and carbohydrate content and structure (Elliott et al. 1997). Ithas been shown that C5 (and hCG from other choriocarcinoma subjects)differ in the protein moiety mainly by the presence of an increasednumber of nicked sites and by increased glycosylation relative to thehCG of normal pregnancy. In comparison with the hCG of normal pregnancy,choriocarcinoma derived hCG has increased fucosylation of the N-linkedbiantennary oligosaccharides in the beta subunit. In addition, the O-linked oligosaccharides in preparation C5 (a form of hCG produced froma single patient with choriocarcinoma) has a 100% tetrasaccharide coreon the COOH-terminal region of the beta subunit. Normal mid pregnancyhCG has only 10-20% of this structure (Elliott et al. 1997). Theseobservations, plus our own determination that the hCG synthesized by theJAR choriocarcinoma cell line provides a B152/B109 isoform ratio similarto that observed in early pregnancy, leads us to the conclusion that invery early pregnancy, the developing trophoblast secretes an isoform ofhCG which resembles that produced in choriocarcinoma.

We have also tested recognition of pituitary hCG since its N-Asncarbohydrates differ somewhat from those of placental hCG, bearing acloser resemblance to those of hLH which have both sialic acid andsulfate groups (Birken et al. 1996). The carbohydrate structure of the bCOOH-terminal portion of pituitary hCG is not yet known. Since B152 didnot recognize any substantial differences between pituitary andplacental hCG (FIG. 3), differences in N-Asn recognition are unlikely.In terms of the COOH-terminal carbohydrates, it appears that pituitaryand placental hCG (mid-pregnancy isoforms) may be similar, assuming theO-linked carbohydrate on the C5 antigen is part of the epitope of B152.

EXAMPLE 2 B152/B109 Ratio Predicts Pregnancy Outcome

The B152/B109 Ratio Measured in Urine Samples Throughout the Pregnancy

The relative concentrations of hCG isoforms in 103 normal pregnancyurine samples (5-39 weeks post last menstrual period—LMP) weredetermined by two immunometric assays (B152-B207* and B109-B108*). Bothbecause of the wide range of hCG concentrations in different samples,even at the same gestational age, and because neither of the assays istotally specific for the two (or more) families of hCG isoforms present,we find that presenting the data as a ratio of the observed two isoformgroups more clearly delineates the change in isoform content aspregnancy progresses. This calculated ratio is shown in FIG. 4. In weeks5-8 of pregnancy, the ratio of B152/B109 isoforms ranged between 6.2 and1.3, indicating a predominance of the B152 isoform(s) in earlypregnancy. During the 10 to 12 week period, the ratio ranged from 1-0.2, indicating that an inversion in hCG isoform content is occurring aspregnancy progresses. This decline in the ratio continues, ranging from0.54-0.08 in the 15-18 week period and reaching an inflection point at29 weeks. At that time, the ratio reached a value of around 0.06 afterwhich the ratio displayed a rise to a range of 0.2 -0.07 in the 37-39.5weeks of gestation time period.

Statistical analysis involved fitting the log transformed ratio data tosecond and third order polynomial regression models. Since the thirdorder term was not significant (likelihood ratio C²(1)=1.32, P=0.25),the second order model was used (r²=0.793). The log B152/B109 ratioreached an inflection point at LMP=29 weeks, based on this model.

The B152-B207* values reflect a measurement of the B152 isoform in termsof later pregnancy hCG equivalents, not in absolute quantities. It mustbe emphasized that the “absolute” concentrations measure in the B152assay cannot be compared with the results of the B109 assay on anequimolar basis since the potency of the hyperglycosylated isoform ismuch higher in the B152 assay vis-a-vis the standard, i.e. normal laterfirst trimester pregnancy hCG. The actual molar values of this isoformare on the order of tenfold less than those recorded in the assay. Forthis reason we have chosen not to analyze absolute molar quantities ofthe two analytes but only the ratio of the two measurements.

Even in normal pregnancy, the hCG values obtained vary widely accordingto the characteristics of the immunological reagents employed (Cole andKardana, 1992; Cole et al. 1993). We hypothesize that the two assaysdescribed in this report primarily detect hCG isoforms at opposing endsof this spectrum, each primarily recognizing a subset of closely relatedmolecules in the continuum of early to later pregnancy hCG molecularforms.

We have retained the use of normal pregnancy hCG as the standard inB152-B207* assay, despite its decreased affinity in this antibodyconfiguration. The reasons for this include the limited and unrenewablesupply of C5 (which was isolated from the urine of a single patient) andthe variability in data which would result from investigations usingdifferent standards. The consequences of this choice are that the earlypregnancy hCG isoforms have markedly increased immunopotency over thatof normal pregnancy and hence their molar quantities are overestimatedin this assay. We use this difference in affinity to our advantage byemploying a ratio of the molar results of two assays (B152 and B109).Either assay taken alone obscures this change due to the wide excursionof hCG values which occur in normal pregnancy.

Others have documented progressive changes in hCG isoforms throughoutpregnancy. Skarulis et al. found that the fucose content of both intacthCG and also its free beta subunit increased as pregnancy progressed(Skarulis et al. 1992). Diaz-Cueto et al. investigating the isoelectricfocusing pattern of circulating hCG throughout pregnancy, found that inearly pregnancy, more than 80% of the hCG isoforms were acidic. Thisfraction decreased to less than half (47%) late in the third trimester(Diaz-Cueto et al. 1996). In contrast, Wide and Hobson found that thehCG of early pregnancy was more “choriocarcinoma-like” by virtue of itsgreater biological activity than the hCG of normal pregnancy (Wide andHobson, 1987). Fein et al., in a study which employed gel filtrationdetermined that first trimester hCG was a larger size than that of thethird trimester. Treatment with exoglycosidases eliminated the sizedifferential, indicating that the first trimester hCG was more highlyglycosylated (Fein et al. 1980).

The B152/B109 Ratio in Matched Serum/Urine Samples in the First andThird Trimesters of Pregnancy Compared With hCG From JAR Cells.

The B152/B109 ratio in serum is analogous to that found in matched urinesamples and undergoes a similar change as pregnancy progresses (FIG. 5).The B152/B109 ratio in the cell supernatant from JAR cells (achoriocarcinoma derived cell line) was similar to that of earlypregnancy.

The B152/B109 ratios of both serum and urine hCG concentrations aresignificantly higher in the first trimester as compared to the thirdtrimester of normal is pregnancies (Table 2). Significant differencesbetween serum and urine hCG concentration ratios as well as logtransformed ratios in early (5-6 weeks) and late (36-39 weeks) gestationwere evaluated by paired t-tests (Table 3). In both the first and thirdtrimesters, urinary B152/B109 ratios were significantly higher thanserum ratios, indicating that there was a preferential clearance of theB152-recognized isoform into urine, regardless of the relativeconcentrations of the two isoforms. TABLE 2 Analysis of the B152/B109ratio in serum and in urine in the first vs third trimesters ofpregnancy. Measure T-test(df) P Serum, ratio t(11) = 6.65 0.0001B152/B109 Serum, log  t(23) = 21.61 0.0000 (ratioB152/B109) Urine, ratiot(11) = 4.64 0.0007 B152/B109 Urine, log t(15.7) = 16.85 0.0001(ratioB152/B109)

TABLE 3 Analysis of the B152/B109 ratio in serum vs urine in the firstand third trimesters of pregnancy. Gestational Paired-t age Measure (df)P 5-6 weeks Ratio B152/B109 t(11) = 3.25 0.0077 Log(ratioB152/B109)t(11) = 6.25 0.0001 36-39 weeks  Ratio B152/B109 t(10) = 5.47 0.0003Log(ratioB152/B109) t(10) = 7.14 0.0001

The B152/B109 Ratio in Urine Samples From IVF Patients

In urine samples from IVF patients (1-4 weeks post embryo transfer—ET)the B152/B109 ratio was again between 2-8 and decreased as pregnancyprogressed (FIG. 6), similar to that observed in natural conceptions.The effect of pregnancy duration with respect to outcome variables couldbest be represented by a linear or quadratic function. ANCOVA modelsincluding the second order week were fitted to the general equation:Outcome=(effect of time post ET)+(effect of diagnosis). After anappropriate ANCOVA model was determined, the least square means(adjusted for week post ET effect) were compared among the normalpregnancy, ectopic pregnancy and spontaneous abortion populations (Table4). The log transformed values of both B109-B108* and B152-B207*measured hCG forms discriminated both ectopic pregnancy and spontaneousabortions from normal pregnancy (P=0.0001). The ratio of the logtransformed values discriminated abortion from normal pregnancy(P=0.016). However, neither the ratio of B152/B109 nor the log of thatratio discriminated either of the pregnancy disorders from normalpregnancy.

A significant number of spontaneous abortions and ectopic pregnanciesoccur in IVF pregnancies. We did not find a difference in the ratio ofthe isoforms between either of these two categories as compared tonormal controls, possibly a consequence of low statistical power.However a significant difference was found between the B152 hCG isoformslevels in normal pregnancy and spontaneous abortion. This supports ourprevious finding in early pregnancy loss, where diminished or absentlevels of the B152 isoforms characterized an early pregnancy loss(O'Connor et al. 1998). TABLE 4 IVF patients: analysis of covariance ofhCG isoforms among normal pregnancy (np), ectopic pregnancy andspontaneous abortion as a function of gestational age. ^(c)Adjusted^(d)-Pairwise Outcome R² ^(e)-F P Difference ^(a)Log (ratio 0.51 0.890.41 none B152/B109) ^(a)Log 0.56 21.33 0.0001 np vs (B109-B108*)abortion & ectopic ^(b)Log (B152)/ 0.45 4.34 0.016 np vs log (B109)abortion ^(b)Log 0.50 26.94 0.0001 np vs (B152-B207*) abortion & ectopic^(a)ANCOVA model with 2nd order polynomial coefficient (or parameter).^(b)ANCOVA model with only 1st order (linear) coefficient.^(c)Adjusted R² is a R² adjusting number of coefficients on the ANCOVAmodel so that comparisons of two R² with different ANOVA models withdifferent number of coefficients are meaningful.^(d)-“Pairwise difference” is based on t-test comparing the least-squaremeans of outcome variables (after adjusting effect of week ET).^(e)-Degree of freedom (df1, df2) for F-test are (2, 82) for a modelwith only linear coefficient and (2, 81) for a model with both linearand 2nd order coefficient.

HCG Analysis of Trophoblastic Disease Samples

Trophoblast disease serum (17 samples) and urine (28 samples) wereobtained from patients post therapy and hence contained low hCG levels.Due to limited amounts of sample all of these specimens were run at a1:10 initial dilutions. HCG levels in serum were low. The highest hCGconcentration in serum was 202 fmol/ml in the B152-B207* assay, with acorresponding value of 148 fmol/ml in the B109-B108* determination. Sixof seventeen samples in serum had detectable levels, with 4/6 having ahigher value in the B152-B207* assay. Of the 15/28 positive iurinesamples however, 14/15 had higher levels in the B152-B207* assay than inthe B109-B108* assay, with the highest hCG value being 20000 fmol/ml inthe B152-B207* assay and 18715 fmol/ml in the corresponding B109-B108*assay. Due to the small sample size, no statistical treatment wasperformed on this data, but even in these post-treatment patients theB152/B109 ratio was >1, which corresponds to the early pregnancy hCGisoform ratio.

The specimen limitations discussed above precludes our reaching anydefinitive conclusion on the analysis of trophoblastic disease samples.However it appears as might be anticipated that the B152 assay is moresensitive than B109 assay in detecting hCG immunoreactivity in the bloodand in the urine of trophoblastic disease patients, even aftertreatment.

Chromatography of First Week of Gestation Pregnancy Pool. In order todetermine whether the B152-B207* assay recognized other forms of hCGassociated immunoreactivity in addition to the intact hCG molecule,specimens were pooled. FPLC on tandem Superose 12 columns followed byimmunoassay of the fractions for all of the characterized forms of hCGrevealed that only the intact hCG molecule (or hCG free beta subunit)gave a signal in this assay (See FIG. 7). There were no lower molecularweight fragments identified by the B152-B207* assay. The hCG free betaanalyte was measured in urine described in FIG. 2 and was found to makea negligible contribution to over all hCG immunoreactivity in thesespecimens.

Molecules recognized by monoclonal antibody B152 in urine and pituitaryextracts. In order to define the nature of the hCG isoforms recognizedby B152, high resolution gel filtration columns of both pituitaryextracts and postmenopausal urine concentrates were used. The rationalefor use of pituitary extracts is to determine cross-reactive molecules,specifically those which are glycosylated, which are plentiful inpituitary which contains the entire family of glycoprotein hormones,hLH, hTSH, and hFSH as well as free subunits and the pituitary form ofhCG. Two peaks are detected in both of these cases. Only one peak wasdetected in similar studies of pregnancy urine concentrates as describedearlier. In the pituitary, it is likely that the larger molecule ispituitary hCG (70K) while the smaller sized molecule is hLH. Since hLHis present at loox or so as compared to pituitary hCG, the apparentsimilar concentration of immunoreactivity indicates that B152 hasreduced cross-reactivity to hLH as compared to hCG. Likewise, both hCGand hLH occur in postmenopausal urine, again with much more hLH than hCGand the B152 pattern is similar to that of the pituitary extract. Theseresults show that B152 is generally hCG specific except for crossreactivity to hLH (as shown by standard cross-reaction studies in TableI) and that its carbohydrate specificity is both to the protein portionas well as to the carbohydrate moieties of hCG (and to a lesser extentof hLH) since it does not react with the multitude of other glycoyslatedproteins present in the pituitary nor with those in postmenopausal urineexcept for hCG or hLH-related molecules.

Serum and urine specimens were analyzed using two assays, B109-B108* andB152-B207*, which recognize the difference in molecular isoforms of hCG.See Table I. The in vitro bioassay for hLH/hCG is described above. (SeeFIG. 1). The immunometric assay employs 96-well microliter platetechnology. The coating antibody, at a concentration determined toprovide the most satisfactory combination of sensitivity and range, isapplied to the microtiter wells (Immulon IV, Dynatech Laboratories) incarbonate buffer (0.2M, pH 9.5). The plates are incubated with thecoating solution at 4° C., overnight, then aspirated, washed withwashing solution (0.05% Tween, 0.15N NaCl), and blocked with a 1%solution of BSA (three hours at room temperature). The BSA solution isaspirated and the appropriate hCG standards (200μL/well), in buffer B(PBS/0.1% bovine IgG/0.1% sodium azide), or in hCG free serum (Chemicon,Inc.), or hCG free urine, as appropriate to the specimen matrix, andspecimens are added to the wells. The plates are sealed with platesealers, and incubated overnight at 4° C. The controls, specimens, andstandards are then aspirated, the plates washed 5 times with washingsolution, and iodinated detection antibody in buffer B (200 μL/well,100,000 cpm/well) added and incubated overnight at 4° C. The wells areagain aspirated, washed 5 times with washing solution, separated andcounted (Packard Cobra gamma counted). Values are interpolated from asmoothed spline transformation of the count data. This assay procedure,as well as assay validation has been previously reported (O'Connor, J.F., et al., 1988).

Creatinine analysis, when urine values are normalized to creatinine, isperformed in a microtiter plate format following a modification of theTaussky procedure (Taussky, H. H., 1954).

Descriptive statistical and graphical methods are applied to measures ofserum and urine samples from normal healthy pregnancies to identify thedistributions a) between patient first trimester average B152 levels,B109 levels and B152/B109 ratio; b) between patient variability in timeto B152/B109 ratio reaching. 1.00; and c) between patient variability intime to B152/B109 ratio declining by 1/3rd from first trimester maximumlevels. The variability in the timing of the crossover in the ratio ofthese two analytes provides an empirical basis from which to estimatethe value of these markers as biochemical signatures of a viable thirdtrimester fetus.

Comparison of the assay profile of healthy normal pregnancies to thoseof unsuccessful pregnancies from failed IVF implantations, twonon-parametric hypotheses are available: 1) the proportion ofpregnancies in which the B152/B109 ratio falls below 1.00 is nodifferent in healthy normal and unsuccessful IVF pregnancies; 2) theproportion of pregnancies in which the B152/B109 ratio declines by 1/3rdfrom first trimester maximum levels is no different in healthy normaland unsuccessful IVF pregnancies. These hypotheses can be tested as adifference between two proportions. For example, a comparison of week 14vs. week 9, week 13 vs. week 6, week 12 vs. week 5 or week 11 vs. week 4pregnancies to show a reversal of the B152/B109 ratio in healthy normalpregnancies and unsuccessful IVF implantations, respectively. The poweranalyses apply to an outcome defined as the time at which the B152/B109ratio declines by 1/3rd from first trimester maximum levels, althoughthis outcome would necessarily provide earlier detection of pregnancyfailure than the reversal of the B152/B109 ratio. Patterns of resultsless discriminantly different from these indicate a rejection of thedichotomous outcome of B152/B109 ratio reversal as a clinicallymeaningful marker of pregnancy failure.

Alternatively, the same two non-parametric hypotheses can be recast asparametric hypotheses by considering the timing of the biochemicalevents within the assay profile of healthy normal pregnancies andunsuccessful pregnancies from failed IVF implantations: 1) the time atwhich the B152/B109 ratio falls below 1.00 is no different in healthynormal and unsuccessful IVF pregnancies; 2) the time at which theB152/B109 ratio declines by 1/3rd from first trimester maximum levels isno different in healthy normal and unsuccessful IVF pregnancies. Ofcourse, the objective is to provide an empirical basis from whichclinicians may counsel their patients. Thus, it is important to adopt alogistic model for this component of the data analysis. With pregnancysuccess as the outcome, logistic models allow the estimation of the(symmetrical) hypothesis of increase in risk of pregnancy failure foreach additional week where either the B152/B109 ratio has failed todecline by one third from first trimester baseline maximum values or theB152/B109 ratio has failed to become less than 1.00 (measured in weeks).The logistic model enables specification of the time at which resultsindicate a particular pregnancy exceeds an a priori defined likelihoodof failure, given assay data regularly available during pregnancy, andallows incorporation of other risks for pregnancy failure in the samedata analytic framework to assess the relative contribution of threatsto pregnancy loss. The Cox proportional hazard model may be used toexamine predictors of the crossover rates. Mixed effects models can alsoanalyze repeated measures of the B152/B109 ratios taken during entirecycles. These models are particularly useful since they allow inclusionof incomplete and imbalance data (i.e. data with missing values andunequal timing of data collection), to estimate effects of time-varyingcovariates, to model dependency structure of repeated measures and tomodel possible heterogeneity of the ratio measures within eachexperimental group.

B152 hCG isoforms isolated from early pregnancy urine and determinationof their protein and carbohydrate structures. Using the alreadydeveloped scheme of concentration and immunoaffinity extraction ofurine, hCG molecules are isolated from urine collected from women inearly pregnancy for both protein and carbohydrate analyses. According toone approach, molecules are isolated from HPLC fractions, digested withproteases before and after reduction of disulfide bonds, examination ofthe resultant peptides by mass spectrometry and/or sequence analysis,isolation of carbohydrate moieties after glycosidase digestions anddetermination of carbohydrate structures by a combination of specificglycosidases and retention times on specialized anion exchange columnsas compared to know branch-chain oligosaccharide standards. In a similarapproach, the final purification stage for the isolated hCG isoforms isSDS gel electrophoresis. Both protease digests and glycosidase digestsare performed on the blotted and cutout band. This method results ingreater purity of the protein and less artifactual errors due tocontamination by carbohydrates which are not in the purified protein butare derived from outside contaminants.

Carbohydrate compositional analyses and oligosaccharide branched chainidentifications. The MALDI TOF mass spectrometric method may be used toconfirm oligosaccharide structures by using specific glycosidases on theglycopeptides and determining the change in molecular weight as thesugars are digested off the glycopeptide. Only the hCG beta COOH peptidecan be expected to contain O-linked sugar moieties. These are of specialinterest since it is thought that B152 has significant reaction withthis region. The structures of this region can be determined in asimilar fashion using enzymes that specifically release O-linkedglycans. The O-linked structures has been previously examined usingstandard reference pregnancy hCG (Cole, L. A., et al., 1985). TheO-linked branched chain structure are determined by a similar strategyusing the Dionex chromatographic system as well as specific glycosidaseson the C-terminal glycopeptides and Mass Spectrometry. In one study(Elliott, M. M., et al., 1997), these techniques were used to elucidatethe carbohydrate structures of CR series hCG preparations (standardurinary pregnancy hCG) and compared them to the structures of patientsamples such as C5 which was the immunogen employed to generate antibodyB152. It was found that C5 contained significantly more mono andtri-antennary (2× mono and 3× tri-structures than the CR preparations)on the N-Asn residues. It was also found that more tetrasaccharidestructures were on the hCG COOH-terminal peptide O-Serine residues inthe choriocarcinoma hCG isoform than in the CR preparations.

Biological activity and metabolic clearance of hCG isoforms. Biologicalactivity is a function both of molecular structure and half-life in thecirculation, which can be influenced by structure. Alterations incarbohydrate/sialic acid content of the glycoprotein hormones arethought to be responsible for the changes in hCGbiological/immunological activity observed throughout pregnancy. Inaddition, signal transduction at the receptor is influenced by the pI ofthe hCG isoform and the presence or absence of carbohydrate. Thus, it isvaluable to examine both receptor binding and biological activity invitro and, in order to determine the mechanism of action, to distinguishreceptor binding and signal transduction as well as relative potency ofsignal transduction along with in vivo bioactivity determinants such ascirculating half life. Studies, including clearance rates, are performedon B152 hCG isoforms of early successful pregnancy, hCG from thirdtrimester pregnancy, and the reference urinary hCG preparation, CR 127.

EXAMPLE 3 B152 and B151 Immunoreactivity in Non-Trophoblastic Malignancy

With the exception of trophoblastic disease and testicular cancer, hCGis expressed in the blood of about 20t of patients with all other typesof cancer(Hussa, R. O., 1987). HCG beta core fragment in the urine has asignificantly higher level of expression, especially in gynecologicalmalignancy. Since the B152 antibody was developed to a form of hCGproduced in a malignancy, it was of interest to examine the expressionof B152 and nicked hCG immunoreactivity (B151) in non-trophoblasticmalignancy. Accordingly, blood and urine derived from men undergoingchemotherapy for prostate cancer or women for ovarian cancer wereevaluated for the expression of hCG isoforms in plasma and urine. It issignificant that in prostate cancer, B152 hCG immunoreactivity is foundin the blood and urine of prostate cancer patients in instances whenthere is no hCG detected by B109-B108*. In ovarian cancer patientsevaluated, there is evidence of nicked hCG in the blood, even in theabsence of both B109 and B152 immunoreactivity. Neither of the abovegroups demonstrated the presence of hCG immunoreactivity when thestandard pregnancy derived hCG assay was employed. It is reassuring tofind that nicked hCG, the existence of which has been documented byseveral investigators, can be found and reliably measured in a clinicalsetting.

Experimental Discussion

In the course of these studies, a potentially important new signal wasobserved in the urine of women early in pregnancy, namely an epitope ofa form of hCG which may indicate the likely success of carrying apregnancy. Likewise, absence of this signal may indicate that EPL willoccur. Since EPL can be a very sensitive marker of environmental toxins(Hakim, R. B., et al., 1995) and is frequently used as anepidemiological marker of exposure, the finding of this epitope providesa powerful tool for monitoring the safety of the environment. Inaddition, this assay facilitates increasing the success rate of IVFinfertility programs since the predictive value of the new measuringsystem would rapidly indicate successful approaches. Described herein isthe novel and completely unexpected finding that successful pregnanciesdisplay a high content of unique isoforms of hCG that are maintained forthe first few weeks of pregnancy and then rapidly decline as pregnancyprogresses. Based on properties of the immunoassay system, it ishypothesized that these hCG isoforms may be hyperglycosylated. This is astriking observation never reported nor suspected earlier. Carbohydrateanalyses (Elliot, M., 1997) demonstrate that C5 hCG employed asimmunogen for antibody B152, contains two times the monoantennarycontent and three times the tri-antennary content of branch chain sugarsas compared to the CR series of natural pregnancy urinary hCG. Inaddition, the O-linked carbohydrates are mostly tetrasaccharide insteadof disaccharide in C5 as compared to CR 127 hCG. (CR 127 hCG is similarto the WHO preparation, the third international hCG standard, which wasCR 119 hCG, prepared by Canfield. and Birken twenty years ago but stillin use today) (Birken, S., et al., 1991a). B152 recognizes C5 hCG muchbetter than nicked CR127 hCG or non-nicked CR 127 hCG (Birken, S., etal., 1993). In addition, JAR cell type hCG is known to contain a similararray of carbohydrate moieties. It was found to be recognized by B152similar to the early pregnancy isoforms in healthy pregnancies. Theobservation that the hCG isoform produced by JAR cells in culture(B152/B109 ratio) is similar to that found in early pregnancy hCGisoforms supports the hypothesis that the production of a type of hCGwith a particular glycosylation pattern is a prerequisite for a viablepregnancy. This glycosylation pattern is not characteristic of the hCGof later pregnancy.

A variety of pregnancy disorders are testable. One category of patientsconsists of those women who experience a high rate of recurrentabortions. Even in populations with no known fertility problems, thetotal rate of pregnancy loss is 32% (EPL plus clinically recognizedabortion) (Wilcox, A. J., et al., 1988). The risk of recurrent abortionincreases with the number of spontaneous abortions experienced in thepast, reaching an incidence of 32% after three consecutive abortions.(Hill, J. A., and Anderson, D. J., 1990). Probable causes of recurrentspontaneous abortion, comprising genetic, infectious, hormonalimbalance, or immunologic factors can be established in less than 60k ofall spontaneous abortions, leaving 40+% of spontaneous abortions with acompletely unestablished etiology. These facts, taken together withreports establishing that the administration of exogenous hCG can be aneffective therapy in subjects with a history of recurrent spontaneousabortion (Quenby, S., and Farquharson, R. G., 1994; Harrison, R. F.,1985) lends support to the hypothesis that a disproportionate productionof the ineffective isoforms of hCG in early pregnancy is a causal factorin both early pre-clinical loss as well as in spontaneous abortion.

A second category includes women undergoing embryo transfer. Thesepatients provide several distinct advantages: The patients undergoingthis procedure are not treated with crude hCG preparations, makingmeasurement of hCG isoforms easy and decisive since all hCG forms derivefrom the embryo none from any injected hCG preparations. Second, is theopportunity to monitor the nature of the isoforms from day 9 of asuccessful pregnancy. Third, is the ability to obtain large volumes ofurine to purify the early pregnancy isoforms to determine theirstructures. Fourth, since pregnancy loss is from 50% to 70% in thispopulation, the loss can be defined as due to lack of the essential hCGisoform recognized by B152 or due to other causes. Comparison of earlypregnancies in populations of women not undergoing in vitrofertilization procedures with those undergoing embryo implantation can,thus, assess whether pregnancy loss situations present similar ordifferent patterns of hCG isoforms during the process. The mechanism ofpregnancy loss in the general population as compared with the muchhigher rate of embryo loss in IVF programs may be different.Additionally, it has been established that the hCG produced inchoriocarcinoma has differences in carbohydrate structures, sialic acidcontent and biological activity (Wide, L., and Hobson, B., 1987; Elliot,M.,. et al., 1997; Hussa, R. A., 1987). Since B152-B207* assayincorporate monoclonal antibodies raised against an immunogen derivedfrom choriocarcinoma, specimens may be evaluated from patients withgestational trophoblastic disease in order to determine whether theabove assays recognize the hCG produced in these conditions with greatersensitivity and specificity than do assays based on the hCG of normalpregnancy, as is apparently the case for the hCG produced in testicularand ovarian cancer.

There are few reports of changes of carbohydrate content of hCG-relatedmolecules during pregnancy. Blithe and colleagues studied free alphasubunit of hCG whose carbohydrate content differs from that of alphawithin hCG by additional carbohydrate antennae and fucose. Thecarbohydrate of free alpha becomes increasingly complex in terms of morebranches and higher content as pregnancy proceeds. It has also beenreported that the quantity of fucose increased in both hCG and in freealpha as pregnancy proceeded (Skarulis, M. C., et al., 1992). Thus, theliterature indicates increasing content and complexity of carbohydrateof hCG and free alpha subunits. However, immunological data using theB152 monoclonal antibody, implies a progression to simpler carbohydratecontent during pregnancy. Since the beta COOH-region's O-linkedcarbohydrates may be involved in the epitope recognized by B152, it isconceivable that the carbohydrate structures of this region may bealtered in a different pattern from the N-linked glycans studied byBlithe and colleagues (Skarulis, M. C., et al., 1992; Blithe, D. L., andIles, R. K., 1995). Data from Skarulis et al. indicate thatheterodimeric hCG may contain additional fucose but do not provide datathat this late pregnancy hCG becomes hyperglycosylated as does freealpha.

Other studies indicated that the forms of hCG during EPL likely differin biological activity from those hCG isoforms in successful pregnancies(Ho, H.-H., et al., 1997). The in vitro bioassays employed in thosestudies are unsuitable for large-scale studies and are not as reliableas the immunoassays described herein. Furthermore, it is likely that invivo assays may give different results since in vitro and in vivo assayssometimes give completely disparate results. In this case, in vivo andclearance assays are most important in order to identify whether the hCGisoforms are truly more potent in the whole animal and to identify thereasons for the increased potency. Thus in vitro and in vivobioactivities of the early pregnancy isoforms of hCG are highlysignificant.

Carbohydrate differences is a widely accepted explanation for variationsin biological to immunological ratio such as the forms observed byvarious studies of EPL (Ho, H.-H., et al., 1997). Various studies(Grotjan, H. R. J., and Cole, L. A., 1989; Hoermann, R., 1997; Stanton,P. G., et al., 1993; Szkudlinski, M. W., et al., 1995, Thotakura, N. R.,et al., 1994; Szkudlinski, M. W., et al., 1993), have shown that sialicacid differences are an explanation for such heterogeneity in biologicalactivities of glycoprotein hormones. These studies have also confirmedthe dogma that in vitro biological activities can yield the oppositeresults from in vivo studies because of altered metabolic clearancerates in the latter studies. Thus, more acidic (more highly sialylated)forms of gonadotropins are more biopotent in the whole animal because ofprolonged circulating half-lives. The same molecules may appear lesspotent in in vitro assays due to greater acidity, greater negativesialic acid content. Hoermann et al. (Hoermann, R., et al., 1997)demonstrated the exclusion of many of the acidic circulating hormoneforms from the urine, thus, prolonging their half-lives. The pI patternof normal pregnancy as well as trophoblastic cancer hCG in serum isquite different from that of urine. Since the studies described hereinindicate that EPL hCG isoforms have reduced in vitro biologicalactivity, this finding cannot be explained solely by what is known ofbiological activity and sialic acid content. Early pregnancy isoformsrecognized by monoclonal antibody B152 may be more potent in vivo byvirtue of prolonged half-life they may then display increased signaltransduction at the receptor as well. This may be explained by ahyperglycosylated form of hCG which is not hypersiaylated. In this case,the extra sugar portion would help prolong circulating half-life of amore basic pI form of hCG which also has increased in vitro bioactivity.

EXAMPLE 4 Diagnosis of Gestational Trophoblast Disease

An important application of the B152 (early hCG isoform)/B109 (late hCGisoform) ratio analysis described herein above is in the very early (andfacile) diagnosis of gestational trophoblast disease. Examples ofgestational trophopblast disease include choriocarcinoma or hydatidiformmole. In normal pregnancy, the ratio of B152/B109 of the two isoforms ofhCG rapidly decreases, eventually inverting. In gestational trophoblastdisease including choriocarcinoma or hydatidiform mole, the ratio isinitially higher than found in normal pregnancy, but does not diminishduring the course of the apparent pregnancy. This approach provides ahighly sensitive and specific diagnostic marker for gestationaltrophoblast disease.

Other pregnancy disorders in which hCG levels are abnormally high orabnormally low include Down's syndrome or other aneuploid pregnancies,ectopic pregnancy, preeclampsia, and intra-uterine growth retardation.Because the hCG production in these conditions is quantitativelyabnormal compared with normal pregnancy, an altered ratio of the hCGisoforms identified by B152 (early hCG isoform) and B109 (late hCGisoform) can be detected.

Thus, the dual isoform analysis (B152/B109) further provides a methodfor diagnosing pregnancy disorders and gestational trophoblast disease.

Materials and Methods

Hormones

The non-nicked hCG isolated from the CR127 preparation of hCG was usedas a standard in both assays (Birken et al. 1993). The pituitary hCG wasisolated as described (Birken et al. 1996). C5, a 100% nicked hCG havingextra sugars on both N- and O-linked carbohydrate moieties, purifiedfrom the urine of a choriocarcinoma patient (Elliott et al. 1997), wassupplied by Dr. Laurence Cole (Yale University School of Medicine).Although the C5 immunogen used in the development of B152 antibody was100% nicked hCG isoform (i.e. had cleavages in the peptide backbone ofloop 2 of the b subunit) the antibody did not discriminate nicked fromnon-nicked hCG (O'Connor et al. 1998).

The same serial dilutions of non-nicked hCG, pituitary hCG and C5 wereused for binding characterization in hCG assays. Hormone concentrationsof initial stock standards solutions were determined by amino acidanalysis.

Immunoradiometric Assays (IRMA)

The methodology used in the construction and validation of the¹B109-B108* assay has been fully described elsewhere (O'Connor et al.1988). The B152-B207* assay has also been characterized (O° Connor etal. 1998). Both assays were performed with a slight modification of thepublished procedure: the capture antibody was adsorbed onto the wells ofmicrotiter plates (Immulon IV, Dynatech, Chantilly, Va.) by incubating a5 μg/ml solution (B109-B108* assay) or 25 mg/ml solution (B152-B207*assay) in coating buffer (0.2 M bicarbonate, pH 9.5) overnight at 4 C.The coating antibody solution was aspirated, the plates washed (washsolution: 0.9% NaCl, 0.05% Tween 20) and blocked with a 1 solution ofBSA in The first antibody is a capture, the second antibody with steriskis an iodinated detection antibody. PBS with 0.1% sodium azide.Following incubation with the BSA solution (minimum 3 hours at roomtemperature) the blocking solution was removed, the wells again washedwith wash solution and 200 ml/well of the appropriate hCG standards wereadded in phosphate buffer B (PBS with 0.1t bovine gamma globulin and0.1% sodium azide). After overnight incubation at 4 C, the plates wereagain aspirated and washed. The 200 ml (50,000 cpm-100,000 cpm) of¹²⁵¹I-labeled antibody was added to the wells which were again incubatedfor 24 h at 4 C. The tracer was aspirated, the plates washed with washsolution, the individual wells placed in glass tubes and theradioactivity determined in a Packard Cobra gamma counter. Doses weredetermined by interpolation from a smoothed spline transformation of thedata points.

All samples were stored frozen at −20 C prior to assay. Because extremevalues of sample pH may interfere with antibody binding, the urine pHwas adjusted with 1.0M Tris (pH 9.0), 50μl/ml urine prior to assay, sothat the final pH was in the range of 7-7.4 (O'Connor et al. 1988).Intra-assay variation was 6% for both assays, inter-assay variation was12% for B109-B108* and 13% for B152-B207* assays. Sensitivity (leastdetectable dose) defined as +2SD from the zero calibrator, was 1 fmol/mlfor the B109-B108* assay and 2.2 fmol/ml for B152-B207* assay.

Patients Samples

Urine samples from IVF patients were a gift from Dr. L. Cole. Theyincluded spontaneous abortion (n=14, range of gestational age 1.8-4.1weeks from ET- embryo transfer), ectopic pregnancies (n=7, gestationalage 2.3-4 weeks) and normal pregnancy controls (n=65, encompassing therange 0.6 to 5.4 week from ET). Some of the normal pregnancy urinesamples throughout the pregnancies were also obtained from Dr. Cole.Others were obtained from the clinical practice of collaboratingphysicians at Columbia Presbyterian Medical Center (CPMC) (Total n=103).Matched serum/urine samples from the first (n=12) and the third (n=11)trimesters were provided by Dr. Amalia Kelly at CPMC. Trophoblastdisease serum (n=17) and urine (n=28) samples were obtained from Dr.Cole, but were collected by Dr. Edward Newlands (Charing Cross Hospital,London, UK). All specimen. collection protocols were approved by theappropriate Institutional Review Board.

Statistical Analysis

Polynomial regression models of log transformed hormone ratios were usedto describe the relationship between the change in ratio as a functionof gestational age in normal pregnancy. A paired t-test was used toevaluate the relationship between matched serum and urine hormoneratios. Analysis of covariance (ANCOVA) was used to describe the timeadjusted relationship of hormone values in ectopic pregnancy andspontaneous abortion to those of normal pregnancy.

Urine Processing.

Twenty-four hour urine samples are collected from women undergoingembryo transfer as well as women in early natural pregnancy. The urineis refrigerated during the collection procedure. After delivery of theurine to the laboratory, sodium azide is added to ig/liter. Womenundergoing in vitro embryo transfer are not pre-treated with hCG. Thus,all hCG which appears in their blood or urine is derived from the embryo(except for the small amounts of pituitary hCG present in all people).Raw urine is freed from particles by centrifugation followed by Pelliconfiltration through a 0.45 micron membrane. Next, the procedure is toconcentrate the urine with a Pellicon (Millipore) system whichconcentrates as much as 30 liters to 500 ml overnight (4° C.) using a3,000 MW cutoff membrane. Smaller volumes can be concentrated in lessthat two hours. Next, the urine is desalted and delipidated by passagethrough a large volume of Sephadex G25 in 0.1 M ammonium bicarbonate.This step greatly increases the binding of CG to immunoaffinity columns.The desalted urinary concentrate is next size fractionated on thePharmacia HiLoad Superdex 200 and the hCG and hCG subunit peaks areidentified by specific immunoassays (O'Connor, J. F., et al., 1994) andthe appropriate fractions are pooled and dried. The hCG and hCG subunitsare purified from the gel filtered urine concentrate by immunoaffinityon insolubilized hCG antibody columns as described but with the use ofeither 4M guanadine (0.1M tris acetate, pH 5) or ammonium thiocyanate aseluant to decrease loss of sialic acid from the hormone. Alternatively,hCG is purified by conventional chromatographic procedures, anionexchange and hydrophobic chromatography. The subunits are separated onreverse phase HPLC using a 0.01M sodium phosphate, pH 5 buffer andacetonitrile, after incubation in 4M guanadine, 0.1M tris acetate, pH 5.A third method is final purification and separation of the hCG subunitson SDS PAGE electrophoresis followed by electroblotting to PVDF. ThePVDF band can be subjected to protease digestion to release peptides andglycopeptides which can be separated on reverse phase HPLC in neutral pH5 buffers.

Separation of Glycopeptides from Isolated hCG Subunits.

To facilitate isolation of the glycopeptides from the hCG subunits, thesubunits are both tryptic digested and the products of digestion areseparated on reverse phase HPLC (using a pH 5 buffer). This procedureresults in removal of the large beta COOH-terminal peptide whichcontains O-linked sugars. It also releases small, non glycopeptides fromboth subunits (Pollak, S., et al., 1990, Birken, S., et al., 1987;Birken, S., et al., 1986). Next, the main disulfide-linked core of eachhCG subunit, is reduced and carboxymethylated, and separated on reversephase HPLC at pH 5. At this stage, large peptides are isolated,including the glycopeptides. Each separated glycopeptide is redigestedwith trypsin and re-separated on HPLC at pH 5. These glycopeptides arenext employed for two different methods of sugar chain analysis. Onemethod is the approach of releasing the oligosaccharides by enzymaticdigestions uing PNGase F for the N-linked glycans. The released glycanscan be separated from the peptides by ethanol precipitation, desialyatedwith neuraminadase, and separated directly on a Dionex Carbopac PA-100column. Oligosaccharide standards are available from Dionex, OxfordGlycosystems and other companies for calibrating column elution timesfor various glycans (Hardy, M. R., and Townsend, R. R., 1994, Rohrer, J.S., et al., 1993, Weitzhandler, M., et al., 1993; Townsend, R. R., etal., 1989). Confirmation of the released structures is obtained byperforming carbohydrate compositional analysis of eluted glycan peaks aswell as performing digestions with specific glycosidases andrechromatographing the modified glycan on the Dionex system (Hardy, M.R., and Townsend, R. R., 1994; Rohrer, J. S., et al 1993; Weitzhanlder,M., et al., 1993; Townsend, R. R., et al., 1989; Townsend, R. R., etal., 1991; Townsend, R. R., et al., 1989; Hardy, M. R., and Townsend, R.R., 1989; Townsend, R. R., et al., 1988; Hardy, M. R., et al, 1997;Hardy, M. R., and Townsend, R. R., 1988; Dionex, 1997; Spellman, M. W.,1990; Kumarasamy, R., 1990). The newly modified glycan can be observedto elute at the same time as the appropriate standard oligosaccaharideand, in addition, the released monosaccharide can frequently beidentified as well (Dionex, 1997). Structure determination isfacilitated by the use of specific glycosidases for branch chaincleavage as well as for digestion of individual sugars from each of thebranch chains. For example, Endo H cleaves high mannose type and hybridoligosaccharide chains while glycosidase Endo F2 cleaves biantennarycomplex types and PNAase F cleaves tri and tetra-antennary chains downto the N-Asn bond.

Competitive Receptor Binding and In vitro Bioassay.

Bioassays are performed with recombinant-engineered CHO cellstransfected with the human receptor to LH/CG Cells are maintained inHam's F-12 medium, 4 mM Glutamine, 400 ug/ml G418 (Gibco) , 5% fetalcalf serum, 100IU/ml penicillin, 100 ug/ml streptomycin. The cells areremoved from the flask surface by versene only.

A competitive receptor assay constructed as follows: The receptorbinding assay mixture contains 100 ul of the appropriate dilution ofserum/urine samples or hCG dilutions for standard curve, 100 ul of¹²⁵-I-hCG (50,000-100,000 cpm) in buffer A(PBS/0.l%BSA) and 100 ul ofCHO cells (2×10⁵ cells in PBS). The mixture is incubated at 37° C. withslight shaking followed by centrifugation for 10 minutes at 750 xg. Thesupernatant is aspirated and the cell pellet is counted ingamma-counter.

In vitro bioassay. Transfected CHO cells are seeded (200,000 cells/well)into a 24 well plate in culture medium and incubated for 2-3 days untilthe cells reach confluence. Non-transfected CHO cells are included tomonitor non-specific response. The medium is removed and replaced withmedium containing 1 mM isobutylmethylxanthine with appropriate dilutionsof tested serum or urine. The plates are incubated at 37° C. for twohours. The supernatant is removed, and the wells washed with Hank'sbalanced salt solution. The intracellular cAMP is extracted with 95%ethanol, which is diluted 1:5, (or up to 1:40, depending on cAMPcontent) in assay buffer provided by the cAMP kit (New England Nuclear).cAMP assay is performed according to manufacturer's instructions.Response is normalized to well protein content (BCA protein assay kit,Pierce, Rockford, Ill.).

In vivo bioassay is determined by the uterine weight assay in immaturefemale mice, following the procedure of Wide and Hobson (Wide, L., andHobson, B., 1987). The mice are injected subcutaneously with one thirdof the total dose of gonadotropin on three consecutive days and killed72 hours after the first injection. Uteri are dissected free frommesentery, fat and oviducts, blotted to remove intrauterine fluid andweighed to the nearest 0.1 mg. Five to ten mice are used at each ofthese dose levels. The hCG standard preparation used is a nicked hCG.This material may be run concurrently with specimens isolated from firstand third trimester pregnancy. Sham saline injection may be used as acontrol. The response signal is the log mouse uterine weight.

Clearance of hCG isoforms. The clearance of hCG is determined in therat. Blood (200 ul/sample) is obtained at 0, 120, 240, 360 and 480minutes post injection, from an indwelling catheter in an catheterizedexternal jugular vein, following the procedure described by Newman etal. (Newman, C. B., et al., 1985) and Brown and Hedge (Brown, M. R., andHedge, G. A., 1972). Briefly, adult male Sprague-Dawley rats (CharlesRiver Laboratories, Wilmington Mass.), wt 175-225 g, are given freeaccess to food and water. Rats are handled for acclimatization for oneweek after arrival, and several days before the hCG infusion, the ratsare cannulated under pentobarbital anesthesia. A 21 gauge stainlesssteel cannula is inserted into the one external jugular vein. Theplacement of the catheter allows for the collection of blood from theunrestrained, unstressed rat. After the animals have recuperated fromthe cannula implacement , an hCG isoform is injected (10 μg/ml sterilesaline) through the cannulated vein. Blood samples are obtained at thefour time intervals listed above. The blood is allowed to clot and theserum separated and stored at −80° C. for immunometric assays specificfor different hCG isoforms.

Clearance rate of the isoforms of hCG from the circulation of the ratare estimated by computer fitting the concentration data to an equationof the general form:

Concentration=Ae^(αt)+Be^(βt) at time t; A and α are parameters of therapid component and β and β are parameters of the slow component. Themetabolic clearance rate (MCR) is calculated as MCR=Dose/(A/α+B/β) andthe initial volume of distribution is calculated from V_(d)=Dose/(A+B).The MCR is normalized to body weight for statistical analysis, which isperformed using ANOVA with Duncan's range test for determination ofsignificance (Cassals, J. W., et al., 1989).

Mice. The mouse species used in the experiments described herein areBalbic mice, aged 12-20 weeks old and adult Sprague-Dawley rats ofeither sex. Mice used for the production of monoclonal antibodiesthrough ascites and for the determination of in vivo biological activityas described. Balbc/c mice are used because hybridoma cell lines weredeveloped using Balb/c splenocytes.

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1. A method of predicting the likelihood of a negative pregnancy outcomein a female subject comprising: a) contacting a sample from the subjectwith a capture antibody which specifically binds to an early pregnancyassociated molecular isoform of hCG under conditions permittingformation of a complex between the antibody and the early pregnancyassociated molecular isoform of hCG; b) measuring the amount ofcomplexes formed, thereby determining the amount of the early pregnancyassociated molecular isoform of hCG in the sample; and c) comparing theamount of early pregnancy associated molecular isoform of hCG in thesample determined in step (b) with the amount determined for atemporally matched, normal pregnant subject, wherein the relativeabsence of the early pregnancy associated molecular isoform of hCG inthe sample indicates a negative outcome of pregnancy for the subject. 2.A method for predicting the likelihood of a negative pregnancy outcomein a female subject comprising: a) contacting a sample from the subjectwith a capture antibody which specifically binds to an early pregnancyassociated molecular isoform of hCG under conditions permittingformation of a complex between the antibody and the early pregnancyassociated molecular isoform of hCG; b) contacting any complex formed instep (a) with a labeled detecting antibody under conditions permittingbinding to the complex the capture antibody and the hCG isoform; c)measuring the amount of labeled detecting antibody bound to the complexso as to thereby determine the amount of the early pregnancy associatedmolecular isoform of hCG in the sample; and d) comparing the amount ofearly pregnancy associated molecular isoform of hCG in the sampledetermined in step (b) with the amount determined for a normal pregnantsubject, wherein the relative absence of the early pregnancy associatedmolecular isoform of hCG in the sample indicates a negative outcome ofpregnancy for the. subject. 3-5. (canceled)
 6. A method of predictingthe likelihood of a negative pregnancy outcome in a female subjectcomprising: a) contacting a sample from the subject with a captureantibody which specifically binds to an early pregnancy associatedmolecular isoform of hCG under conditions permitting formation of acomplex between the antibody and the early pregnancy associatedmolecular isoform of hCG; b) measuring the amount of complexes formed,thereby determining the amount of the early pregnancy associatedmolecular isoform of hCG in the sample; and c) comparing the amountmeasured in step (b) with the amount determined by contacting the samesample with a second capture antibody which specifically binds to intactnon-nicked hCG without substantially cross-reacting with said antibodyunder conditions permitting formation of a complex between the antibodyand the early pregnancy associated molecular isoform of hCG and a seconddetection antibody, wherein a high ratio of amounts determined for saidfirst capture antibody relative to the second capture antibody indicatesa positive outcome of pregnancy for the subject, and a low ratioindicates a negative outcome of pregnancy for the subject. 7-57.(canceled)